The noncanonical NF-?B signaling pathway mediates activation of specific NF-?B members, p52 and RelB, which regulate important biological functions such as osteoclastogenesis, lymphoid organogenesis, lymphocyte development and activation, and generation of immunological tolerance. This novel NF-?B pathway relies on processing of the p52 precursor protein, p100. Since p100 functions as a cytoplasmic inhibitor of RelB, the p100 processing not only generates p52 but also causes nuclear translocation of active p52/RelB NF-?B complex. Over the past few years, we have made seminal pioneer findings demonstrating that the processing of p100 is tightly regulated through its site-specific phosphorylation by a novel kinase complex composed of NIK and IKK1. Our work also reveals the aberrant p100 processing in leukemia T cells transformed by the human T-cell leukemia virus (HTLV). The overall objective of this continuation application is to elucidate the molecular mechanisms mediating normal and deregulated noncanonical NF-?B signaling. The proposed project is based on strong preliminary data and published work from our laboratory. In particular, our recent work suggests a novel mechanism of NIK regulation, which appears to involve its dynamic interaction with a negative regulator, TRAF3, and modulation of its expression level. Intriguingly, induction of noncanonical NF-?B signaling is associated with TRAF3 degradation, although how this intermediate signaling step is regulated remains unclear. Our preliminary studies also reveal the involvement of novel regulators of noncanonical NF-?B signaling. Moreover, we have made significant progress towards understanding the pathological activation of noncanonical NF-?Bs by the leukemia virus HTLV. Based on these findings, we hypothesize that noncanonical NF-?B signaling is tightly controlled by negative and positive regulators, the deregulation of which may contribute to both immunological disorders and HTLV-induced T-cell malignancies. We will perform three specific aims to examine our hypotheses. (1) Elucidate the biochemical mechanisms that regulate the signaling function of NIK. (2) Characterize the intermediate signaling steps and molecular components of the noncanonical NF-?B pathway. (3) Investigate the role of noncanonical NF-?B pathway in normal and pathological T-cell activation. PUBLIC HEALTH RELEVANCE: The NF-?B family of transcription factors regulates diverse biological processes, most notably immune and inflammatory responses. The focus of this research project is to understand a noncanonical (or atypical) signaling pathway of NF-?B activation. This pathway leads to activation of a sub-group of NF-?B members and is required for specific adaptive immune functions, including lymphoid organ formation, lymphocyte development and activation. Uncontrolled noncanonical NF-?B signaling is linked to chronic inflammation and autoimmunity, whereas defect in this pathway causes immune deficiencies. The studies proposed in this application address the molecular mechanisms of noncanonical NF-?B signaling and will be important for the development of new and effective immune therapies.